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Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR
Jeong-soon Kim, Nian Wang
BMC Research Notes , 2009, DOI: 10.1186/1756-0500-2-37
Abstract: We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR) indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA.Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.Citrus Huanglongbing (HLB) is one of the most devastating diseases on citrus and is associated with a phloem limited bacterium which has yet to be cultured in vitro. Consequently, the pathogen was given a provisional Candidatus status in nomenclature [1,2]. Currently, three species of the pathogen are recognized from trees with HLB disease based on 16S rDNA sequence: Candidatus Liberibacter asiaticus (Las), Ca. Liberibacter africanus (Laf), and Ca. Liberibacter americanus (Lam); Las is the most prevalent species among HLB infected trees [1,3-5]. Las has been spreading worldwide over the last century and has been identified in Japan, China, Southeast Asia, India, Arabian Peninsula, Br
Predictive Sequence Analysis of the Candidatus Liberibacter asiaticus Proteome  [PDF]
Qian Cong, Lisa N. Kinch, Bong-Hyun Kim, Nick V. Grishin
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0041071
Abstract: Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a parasitic Gram-negative bacterium that is closely associated with Huanglongbing (HLB), a worldwide citrus disease. Given the difficulty in culturing the bacterium and thus in its experimental characterization, computational analyses of the whole Ca. L. asiaticus proteome can provide much needed insights into the mechanisms of the disease and guide the development of treatment strategies. In this study, we applied state-of-the-art sequence analysis tools to every Ca. L. asiaticus protein. Our results are available as a public website at http://prodata.swmed.edu/liberibacter_as?iaticus/. In particular, we manually curated the results to predict the subcellular localization, spatial structure and function of all Ca. L. asiaticus proteins (http://prodata.swmed.edu/liberibacter_as?iaticus/curated/). This extensive information should facilitate the study of Ca. L. asiaticus proteome function and its relationship to disease. Pilot studies based on the information from our website have revealed several potential virulence factors, discussed herein.
PCR detection of Candidatus Liberibacter asiaticus, the agent of Huanglongbin or greening disease in citrus  [cached]
Indian Phytopathology , 2012,
Abstract: The causal agent of Huanglongbing (HLB) or citrus greening disease (CGD) was detected from total DNA isolated from midrib of leaves of citrus trees from Delhi, Jammu and Maharashtra showing symptoms of yellowing or yellow mottling by PCR amplification of ribosomal protein gene. The amplicon was cloned in pGEM-T easy vector and sequenced. The clone was 703 nucleotide long and showed 100 % nucleotide sequence identity with part of b operon gene of Asian species of greening bacterium i.e. Candidatus Liberibacter asiaticus but differed from African species of greening bacterium i.e. Candidatus Liberibacter africanus. Our studies indicated that b operon gene of Asian species of greening bacterium is highly conserved and can be used for quick detection of greening bacterium in citrus plant for phytosanitary and certification programme.
Molecular characterization of a mosaic locus in the genome of 'Candidatus Liberibacter asiaticus'
Xuefeng Wang, Changyong Zhou, Xiaoling Deng, Huanan Su, Jianchi Chen
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-18
Abstract: A genomic region (CLIBASIA_05640 to CLIBASIA_05650) of 'Ca. L. asiaticus' showing hyper-sequence variation or locus mosaicism was identified and investigated using 262 bacterial strains (188 from China and 74 from Florida). Based on the characteristic electrophoretic profiles of PCR amplicons generated by a specific primer set, eight electrophoretic types (E-types) were identified, six E-types (A, B, C, D, E, and F) in China and four E-types (A, C, G, and H) in Florida. The 'Ca. L. asiaticus' strains from China consisted predominately of E-type A (71.3%) and E-type B (19.7%). In contrast, the 'Ca. L. asiaticus' strains from Florida was predominated by E-type G (82.4%). Diversity of 'Ca. L. asiaticus' in China was also evidenced. Strains from the high altitude Yunnan Province consisted of five E-types with E-type B being the majority (62.8%), whereas strains from the low altitude coastal Guangdong Province consisted of only two E-types with E-type A as the majority (97.0%). Sequence analyses revealed that variation of DNA amplicons was due to insertion/deletion events at CLIBASIA_05650 and the downstream intergenic region.This study demonstrated the genomic mosaicism of 'Ca. L. asiaticus' resulted from active DNA insertion/deletion activities. Analyses of strain variation depicted the significant inter- and intra-continent diversity of 'Ca. L. asiaticus'.Huanglongbing (HLB) is a destructive disease of citrus production worldwide. All known commercial citrus cultivars are susceptible to HLB. The disease was first noted in Chaoshan area in Guangdong Province of the People's Republic of China in the late of 1800s [1] and is currently distributed in 10 citrus producing provinces in South China. HLB is now established in Sao Paulo of Brazil [2] and Florida of the United States [3] where it poses a great threat to the citrus industry. The disease is associated with three species of non-culturable, phloem-limited, α-Proteobacteria: 'Candidatus Liberibacter asiaticus', 'Ca.
Prophage-Mediated Dynamics of ‘Candidatus Liberibacter asiaticus’ Populations, the Destructive Bacterial Pathogens of Citrus Huanglongbing  [PDF]
Lijuan Zhou, Charles A. Powell, Wenbin Li, Mike Irey, Yongping Duan
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0082248
Abstract: Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus’ (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.
The Transcriptional Activator LdtR from ‘Candidatus Liberibacter asiaticus’ Mediates Osmotic Stress Tolerance  [PDF]
Fernando A. Pagliai equal contributor,Christopher L. Gardner equal contributor,Lora Bojilova,Amanda Sarnegrim,Cheila Tamayo,Anastasia H. Potts,Max Teplitski,Svetlana Y. Folimonova,Claudio F. Gonzalez ,Graciela L. Lorca
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1004101
Abstract: The causal agent of Huanglongbing disease, ‘Candidatus Liberibacter asiaticus’, is a non-culturable, gram negative, phloem-limited α-proteobacterium. Current methods to control the spread of this disease are still limited to the removal and destruction of infected trees. In this study, we identified and characterized a regulon from ‘Ca. L. asiaticus’ involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR), and a predicted L,D-transpeptidase (ldtP). In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype) and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of ‘Ca. Liberibacter asiaticus’, using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease.
Convenient Detection of the Citrus Greening (Huanglongbing) Bacterium ‘Candidatus Liberibacter asiaticus’ by Direct PCR from the Midrib Extract  [PDF]
Takashi Fujikawa, Shin-Ichi Miyata, Toru Iwanami
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057011
Abstract: A phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’ (Las) is a major pathogen of citrus greening (huanglongbing), one of the most destructive citrus diseases worldwide. The rapid identification and culling of infected trees and budwoods in quarantine are the most important control measures. DNA amplification including conventional polymerase chain reaction (PCR) has commonly been used for rapid detection and identification. However, long and laborious procedures for DNA extraction have greatly reduced the applicability of this method. In this study, we found that the Las bacterial cells in the midribs of infected leaves were extracted rapidly and easily by pulverization and centrifugation with mini homogenization tubes. We also found that the Las bacterial cells in the midrib extract were suitable for highly sensitive direct PCR. The performance of direct PCR using this extraction method was not inferior to that of conventional PCR. Thus, the direct PCR method described herein is characterized by its simplicity, sensitivity, and robustness, and is applicable to quarantine testing.
Transcriptional and Microscopic Analyses of Citrus Stem and Root Responses to Candidatus Liberibacter asiaticus Infection  [PDF]
Valente Aritua, Diann Achor, Frederick G. Gmitter, Gene Albrigo, Nian Wang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0073742
Abstract: Huanglongbing (HLB) is the most destructive disease that affects citrus worldwide. The disease has been associated with Candidatus Liberibacter. HLB diseased citrus plants develop a multitude of symptoms including zinc and copper deficiencies, blotchy mottle, corky veins, stunting, and twig dieback. Ca. L. asiaticus infection also seriously affects the roots. Previous study focused on gene expression of leaves and fruit to Ca. L. asiaticus infection. In this study, we compared the gene expression levels of stems and roots of healthy plants with those in Ca. L. asiaticus infected plants using microarrays. Affymetrix microarray analysis showed a total of 988 genes were significantly altered in expression, of which 885 were in the stems, and 111 in the roots. Of these, 551 and 56 were up-regulated, while 334 and 55 were down-regulated in the stem and root samples of HLB diseased trees compared to healthy plants, respectively. Dramatic differences in the transcriptional responses were observed between citrus stems and roots to Ca. L. asiaticus infection, with only 8 genes affected in both the roots and stems. The affected genes are involved in diverse cellular functions, including carbohydrate metabolism, cell wall biogenesis, biotic and abiotic stress responses, signaling and transcriptional factors, transportation, cell organization, protein modification and degradation, development, hormone signaling, metal handling, and redox. Microscopy analysis showed the depletion of starch in the roots of the infected plants but not in healthy plants. Collapse and thickening of cell walls were observed in HLB affected roots, but not as severe as in the stems. This study provides insight into the host response of the stems and roots to Ca. L. asiaticus infection.
Multilocus microsatellite analysis of 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing worldwide
Md-Sajedul Islam, Jonathan M Glynn, Yang Bai, Yong-Ping Duan, Helvecio D Coletta-Filho, Gopal Kuruba, Edwin L Civerolo, Hong Lin
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-39
Abstract: A panel of seven polymorphic microsatellite markers was developed for 'Ca. L. asiaticus'. Microsatellite analyses across the samples showed that the genetic diversity of 'Ca. L. asiaticus' is higher in Asia than Americas. UPGMA and STRUCTURE analyses identified three major genetic groups worldwide. Isolates from India were genetically distinct. East-southeast Asian and Brazilian isolates were generally included in the same group; a few members of this group were found in Florida, but the majority of the isolates from Florida were clustered separately. eBURST analysis predicted three founder haplotypes, which may have given rise to three groups worldwide.Our results identified three major genetic groups of 'Ca. L. asiaticus' worldwide. Isolates from Brazil showed similar genetic makeup with east-southeast Asian dominant group, suggesting the possibility of a common origin. However, most of the isolates recovered from Florida were clustered in a separate group. While the sources of the dominant 'Ca. L. asiaticus' in Florida were not clearly understood, the less-pervasive groups may have been introduced directly from Asia or via Brazil. Notably, the recent outbreak of HLB in Florida probably occurred through multiple introductions. Microsatellite markers developed in this study provide adequate discriminatory power for the identification and differentiation of closely-related isolates, as well as for genetic studies of 'Ca. L. asiaticus'.Huanglongbing (HLB) is one of the most devastating diseases of citrus, which is characterized by the development of yellow shoots and stunted growth of infected trees combined with a decline in quantity and quality of fruit production [1]. HLB-affected fruit are abnormally-pigmented, developmentally flawed, and have a bitter taste- making them unusable for juice production or as table fruit [2,3]. Typically, trees with HLB succumb to the effects of infection and die within a few years after showing the first signs of the disease [4].HL
Deciphering the Bacterial Microbiome of Citrus Plants in Response to ‘Candidatus Liberibacter asiaticus’-Infection and Antibiotic Treatments  [PDF]
Muqing Zhang, Charles A. Powell, Lesley S. Benyon, Hui Zhou, Yongping Duan
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0076331
Abstract: The bacterial microbiomes of citrus plants were characterized in response to ‘Candidatus Liberibacter asiaticus’ (Las)-infection and treatments with ampicillin (Amp) and gentamicin (Gm) by Phylochip-based metagenomics. The results revealed that 7,407 of over 50,000 known Operational Taxonomic Units (OTUs) in 53 phyla were detected in citrus leaf midribs using the PhyloChip? G3 array, of which five phyla were dominant, Proteobacteria (38.7%), Firmicutes (29.0%), Actinobacteria (16.1%), Bacteroidetes (6.2%) and Cyanobacteria (2.3%). The OTU62806, representing ‘Candidatus Liberibacter’, was present with a high titer in the plants graft-inoculated with Las-infected scions treated with Gm at 100 mg/L and in the water-treated control (CK1). However, the Las bacterium was not detected in the plants graft-inoculated with Las-infected scions treated with Amp at 1.0 g/L or in plants graft-inoculated with Las-free scions (CK2). The PhyloChip array demonstrated that more OTUs, at a higher abundance, were detected in the Gm-treated plants than in the other treatment and the controls. Pairwise comparisons indicated that 23 OTUs from the Achromobacter spp. and 12 OTUs from the Methylobacterium spp. were more abundant in CK2 and CK1, respectively. Ten abundant OTUs from the Stenotrophomonas spp. were detected only in the Amp-treatment. These results provide new insights into microbial communities that may be associated with the progression of citrus huanglongbing (HLB) and the potential effects of antibiotics on the disease and microbial ecology.
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